Method of producing 2′-fucosyllactose using recombinant Corynebacterium glutamicum

ABSTRACT

Provided is a method of producing 2′-fucosyllactose including culturing in a medium supplemented with lactose a recombinant  Corynebacterium glutamicum  transformed to express α-1,2-fucosyltransferase, transformed to express GDP-D-mannose-4,6-dehydratase, transformed to express GDP-L-fucose synthase, and transformed to express lactose permease, wherein the recombinant  Corynebacterium glutamicum  has phosphomannomutase and GTP-mannose-1-phosphate guanylyltransferase.

SEQUENCE LISTING

The Sequence Listing submitted in text format (.txt) filed on Oct. 1,2019, named “SequenceListing.txt”, created on Oct. 1, 2019, 12.9 KB, isincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to recombinant Corynebacterium glutamicum(C. glutamicum) which is transformed to expressα-1,2-fucosyltransferase, GDP-D-mannose-4,6-dehydratase (Gmd),GDP-L-fucose synthase(GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase, WcaG) and lactosepermease (LacY), and to overexpress phosphomannomutase (ManB) andGTP-mannose-1-phosphate guanylyltransferase (ManC), and a method forproducing fucosyllactose using the same.

BACKGROUND ART

Human breast milk contains 200 or more kinds of human milkoligosaccharides (HMOs) having a unique structure at a considerablyhigher concentration (5 to 15 g/L) than other mammal's breast milk.

Breastfeeding during infancy is considerably important since HMOsprovide various biological activities that have positive influences oninfant development and health, such as prebiotic effects, prevention ofpathogen infection, regulation of the immune system, and braindevelopment.

Breast milk contains about 200 kinds of oligosaccharides. Among them,2′-fucosyllactose and 3′-fucosyllactose are reported to be main HMOsthat are involved in various biological activities. For this reason, inrecent years, fucosyllactose draws a great deal of attention because ithas potential to be used for powdered milks for infants, healthfunctional food materials for elderly people and medicinal materials.However, it is known that about 20% of women cannot synthesizefucosyllactose well due to mutation of fucose transferase thatsynthesizes fucosyloligosaccharide. For this reason, there is a need forindustrial production of fucosyllactose.

However, since industrial mass-production of fucosyllactose is difficultat present, instead of fucosyllactose, galactooligosaccharide orfructooligosaccharide, which is an analogue of fucosyllactose, is addedto baby food, to offer similar effects thereto.

Meanwhile, methods of producing fucosyllactose include direct extrusionfrom breast milk, and chemical or enzymatic synthesis.

Direct extraction has drawbacks of limited breast milk supply and lowproductivity. Chemical synthesis has drawbacks of expensive substrates,low stereo-selectivity and production yield, and use of toxic organicsolvents. In addition, enzymatic synthesis has drawbacks in thatGDP-L-fucose used as a donor of fucose is very expensive andpurification of fucosyltransferase involves high costs.

Due to the aforementioned drawbacks, it is difficult to apply directextraction, and chemical or enzymatic production to mass-production offucosyllactose and there are almost no technologies for mass-production.However, since it is possible to expect development of functional healthfoods and medicinal materials using 2′-fucosyllactose, a great deal ofresearch is needed for industrial production of 2′-fucosyllactose usingmicroorganisms.

In addition, the majority of conventional methods for producing2′-fucosyllactose using microorganisms were production using recombinantEscherichia coli. However, most Escherichia coli used forexperimentation are predominantly known to be harmful to customersalthough they are not pathogens.

In addition, since an ingredient for the cell membrane of Escherichiacoli may serve as endotoxin, high isolation and purification costs areinvolved in the production of 2′-fucosyllactose. Accordingly, there is adifficulty in using Escherichia coli as a host cell that producesfucosyllactose which is one of food and medicinal materials.

DISCLOSURE Technical Problem

Therefore, the present invention has been made in view of the aboveproblems, and it is one object of the present invention to develop andprovide a method for producing 2′-fucosyllactose at a highconcentration, a high yield and a high productivity while using, as ahost cell that produces fucosyllactose which is a food and/or medicinalmaterial, Corynebacterium glutamicum that is safer than Escherichiacoli.

Technical Solution

In accordance with the present invention, the above and other objectscan be accomplished by the provision of recombinant Corynebacteriumglutamicum which is transformed to express α-1,2-fucosyltransferase, istransformed to express GDP-D-mannose-4,6-dehydratase, is transformed toexpress GDP-L-fucose synthase, and is transformed to express lactosepermease, wherein the recombinant Corynebacterium glutamicum hasphosphomannomutase and GTP-mannose-1-phosphate guanylyltransferase.

The prevent inventors obtained a patent regarding a method for producing2′-fucosyllactose using Escherichia coli, as Korean Patent No.10-1544184 (2015.08.21). However, it has been frequently indicated thatproducing 2′-fucosyllactose for functional food additive applicationsusing Escherichia coli may cause problems due to varioussafety-associated risks associated with Escherichia coli. Accordingly,in accordance with the present invention, there is an attempt to produce2′-fucosyllactose using an alternative strain free of food safetyproblems.

The present invention adopts Corynebacterium glutamicum as a host cellproducing 2′-fucosyllactose. Unlike conventionally used Escherichiacoli, this strain is considered to be a GRAS (generally recognized assafe) strain which does not produce endotoxins and is widely used forindustrially producing amino acids and nucleic acids that are foodadditives. Accordingly, Corynebacterium glutamicum is considered to be astain suitable for production of food and medicinal materials and toadvantageously eliminate customer fears about safety.

However, since Escherichia coli and Corynebacterium glutamicum haveinherently different strain genetic properties, strategies differentfrom Escherichia coli should be applied to Corynebacterium glutamicum.Escherichia coli and Corynebacterium glutamicum are the same in thatexternal α-1,2-fucosyltransferase should be basically incorporated inorder to produce 2′-fucosyllactose. However, Corynebacterium glutamicumfurther requires incorporation of GDP-D-mannose-4,6-dehydratase (Gmd),GDP-L-fucose synthase (this enzyme is called“GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase” and is alsosimply referred to as “WcaG”, and a gene encoding this enzyme isparticularly referred to as “WcaG”), and lactose permease (LacY). Thatis, Escherichia coli has genes encoding GDP-D-mannose-4,6-dehydratase(Gmd), GDP-L-fucose synthase (WcaG), and lactose permease (LacY), butthe Corynebacterium glutamicum strain has no genes encoding theseenzymes, so incorporating such genes from an external source andexpressing the same are needed.

In this case, the genes encoding α-1,2-fucosyltransferase are preferablyderived from Helicobacter pylori, and genes encodingGDP-D-mannose-4,6-dehydratase (Gmd), GDP-L-fucose synthase (WcaG) andlactose permease (LacY) are preferably derived from Escherichia coli.

Meanwhile, the recombinant Corynebacterium glutamicum of the presentinvention is preferably transformed to overexpress phosphomannomutase,and overexpress GTP-mannose-1-phosphate guanylyltransferase.Corynebacterium glutamicum possesses genes encoding phosphomannomutase(ManB) and GTP-mannose-1-phosphate guanylyltransferase (ManC), and canthus express the same. Therefore, there may be no need to incorporategenes encoding the enzymes, but the enzymes should be overexpressed formass-production. For this reason, the present invention requirestransformation of Corynebacterium glutamicum in order to overexpress thetwo enzymes.

Meanwhile, the actions of the enzymes can be seen from FIG. 1 and adetailed explanation thereof is thus omitted. It should be noted thatlactose permease (LacY) is an enzyme involved in transporting lactosepresent outside the strain to the inside thereof. In the followingexample of the present invention, lacYA genes lacZ of which is removedfrom Lac operon in Escherichia coli are incorporated forexperimentation. However, since incorporating Lac operon in the presentinvention aims at incorporating lactose, there is no need to incorporatelacA genes and incorporation of only lacY genes is enough.

Meanwhile, the term “expression” as used herein means incorporation andexpression of external genes into strains in order to intentionallyexpress enzymes that cannot be inherently expressed by theCorynebacterium glutamicum strain according to the present invention,and the term “overexpression” as used herein means overexpression thatis induced by artificially increasing the amount of expressed enzyme inorder to increase expression for mass-production, although theCorynebacterium glutamicum strain according to the present invention hasgenes encoding the corresponding enzyme and therefore can self-expressthe same.

Meanwhile, the present inventors can mass-produce 2′-fucosyllactose,which is breast milk oligosaccharide, in Corynebacterium glutamicum (C.glutamicum) through the transformation strategy described above.

Meanwhile, according to the present invention, genes encodingα-1,2-fucosyltransferase are, for example, fucT2 genes, more preferably,fucT2 genes that have a nucleic acid sequence described in SEQ ID NO: 6,which is obtained by modifying a part of bases of wild-type fucT2 genes(for example, SEQ ID NO: 4). When fucT2 genes having a nucleic acidsequence described in SEQ ID NO: 6 are incorporated, the amount ofproduced 2′-fucosyllactose can be increased, compared to wild-type fucT2genes.

Meanwhile, the present invention provides a method for producing2′-fucosyllactose including culturing the recombinant Corynebacteriumglutamicum of the present invention in a medium supplemented withlactose. When the recombinant Corynebacterium glutamicum strainaccording to the present invention is used, 2′-fucosyllactose can beproduced at a high concentration, a high yield and a high productivity.

Meanwhile, regarding the method for producing 2′-fucosyllactoseaccording to the present invention, the medium preferably furtherincludes glucose. By adding glucose to the medium, growth of strain canbe facilitated and 2′-fucosyllactose can thus be produced at a higherproductivity.

Meanwhile, the method for producing 2′-fucosyllactose according to thepresent invention is preferably carried out by fed-batch culture thatinvolves further supplying glucose or lactose. When glucose or lactoseis continuously supplied by fed-batch culture, cell growth can beimproved and fucosyllactose can be produced at a high purity, high yieldand high productivity. The detailed technologies associated withfed-batch culture are well-known in the art and are not disclosedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 shows a metabolic pathway to bio-synthesize GDP-L-fucose andfucosyllactose in Corynebacterium glutamicum (C. glutamicum) strain;

FIG. 2 is a graph showing effects of incorporation of lacZ-removed lacoperon (lacYA) on production of 2′-fucosyllactose in Corynebacteriumglutamicum (C. glutamicum). FIG. 2A shows results of culture ofCorynebacterium glutamicum (C. glutamicum) strain that overexpress onlyManB, ManC, Gmd and WcaG (Control Group), FIG. 2B shows culture resultsof the strain that overexpresses ManB, ManC, Gmd and WcaG, and allowsfor further incorporation of FucT2 (Comparative Example 1), FIG. 2Cshows culture results of the strain in which lac operon (lacYA), ManB,ManC, Gmd, WcaG, FucT2 and lacZ genes of which are removed, isincorporated (Example 1). Symbols in the graphs have the followingmeanings: ●: dried cell weight, ▪: glucose, ▴: lactose, ▾: lactate, ♦:2′-fucosyllactose;

FIG. 3 is a graph showing results of batch culture using recombinantCorynebacterium glutamicum (C. glutamicum) pVBCL+pEGWT. FIG. 3A showsresults of flask batch culture and FIG. 3B shows results of fermenterbatch culture. When optical density (OD₆₀₀) reaches about 0.8, IPTG andlactose are added to allow final concentrations to become 1.0 mM, and 10g/L (arrows). Symbols in the graphs have the following meanings: ●:Dried cell weight, ▪: Glucose, ▴: Lactose, ▾: Lactate, ♦:2′-Fucosyllactose;

FIG. 4 shows results confirming production of 2′-fucosyllactose throughLC-MS/MS analysis of a batch culture medium solution of recombinantCorynebacterium glutamicum. FIG. 4A is a graph showing production offucosyllactose through molecular weight analysis in a cation mode usingMALDI-TOP MS and FIG. 4B is a graph showing structural composition offucosyllactose identified by tandem mass spectrometry (MS/MS);

FIG. 5 is a graph showing results of fed-batch culture using recombinantCorynebacterium glutamicum (C. glutamicum) pVBCL+pEGWT. After 40 g/Lglucose supplied at an initial stage was completely consumed, glucosestarted to be supplied by a continuous feeding method. At the same time,IPTG and lactose were added (large arrows). Symbols in the graphs havethe following meanings: ●: Dried cell weight, ▪: Glucose, ▴: Lactose, ▾:Lactate, ♦: 2′-Fucosyllactose;

FIG. 6 shows results of codon-optimization of fucT2 genes to be suitedto Corynebacterium glutamicum (C. glutamicum) in order to improvetranslation efficiency of fucT2 genes; and

FIG. 7 is a graph showing effects of incorporation of codon-optimizedfucT2 genes (COfucT2) into Corynebacterium glutamicum (C. glutamicum) onproduction of 2′-fucosyllactose. FIG. 7A is a graph showing results offlask batch culture using recombinant Corynebacterium glutamicum (C.glutamicum) pVBCL+pEGWT (CO). When optical density (OD₆₀₀) reached about0.8, IPTG and lactose were added to allow final concentrations to become1.0 mM, and 10 g/L (arrows). FIG. 7B is a graph showing results offermenter fed-batch culture using recombinant Corynebacterium glutamicum(C. glutamicum) pVBCL+pEGWT (CO). After 40 g/L glucose supplied at aninitial stage was completely consumed, glucose started to be supplied bya continuous feeding method. At the same time, IPTG and lactose wereadded (large arrows). Symbols in the graphs have the following meanings:●: Dried cell weight, ▪: Glucose, ▴: Lactose, ▾: Lactate, ♦:2′-Fucosyllactose.

BEST MODE

Hereinafter, the present invention will be described in more detail withreference to the following examples, and the scope of the presentinvention is not limited to the examples and includes variations oftechnical concepts equivalent thereto.

Example 1: Production of Recombinant Strains and Plasmids

Escherichia coli TOP10 and Corynebacterium glutamicum (C. glutamicum)ATCC 13032 were used to produce plasmids and 2′-fucosyllactose (2′-FL),respectively.

In order to establish pVBCL plasmids, manB genes were amplified throughPCR reaction using two DNA primers (F PstI-manB and RBamHI-SpeI-XbaI-manB) from the genomic DNAs of Corynebacteriumglutamicum ATCC 13032, treated with restriction enzymes PstI and BamHI,and inserted into pVWEx2 plasmids which had been treated with the samerestriction enzymes. manC genes were amplified again by PCR reactionusing two DNA primers (F XbaI-manC and R SpeI-manC) from the genomicDNAs of Corynebacterium glutamicum ATCC 13032, treated with restrictionenzymes XbaI and SpeI, and inserted into the established plasmid toestablish pVmBC. In addition, lacYA gene clusters were amplified by PCRreaction using two DNA primers (F_inf_AsiSI_lacYA and R_inf_AsiSI_lacYA)from the pGlacYA plasmid established in the prior art (Korean Patent No.10-1544184), treated with the restriction enzyme AsiSI and was insertedinto the pVmBC plasmid which had been treated with the same restrictionenzyme, to establish pVBCL plasmids.

In addition, in order to establish pEGWT plasmids, gmd-wcaG geneclusters were amplified by PCR reaction using two DNA primers(F_KpnI-gmd and R_SacI-wcaG) from the genomic DNAs of Escherichia coliK-12 MG1655, treated with restriction enzymes KpnI and SacI, andinserted into the pEKEx2 plasmid which had been treated with the samerestriction enzymes to establish pEGW.

In addition, fucT2 genes were amplified by PCR reaction using two DNAprimers (F_inf_SacI_RBS_fucT2 and R_inf_SacI_fucT2) from the genomicDNAs of Helicobacter pylori ATCC 700392, treated with the restrictionenzyme SacI and inserted into the pEGW plasmid which had been treatedwith the same restriction enzyme to establish pEGWT.

In addition, codon-optimized fucT2 genes (COfucT2) were amplified by PCRreaction using two DNA primers (F_SacI_RBS_COfucT2 and R_SacI_COfucT2)designed from the pBHA (COfucT2) plasmid, and the amplified COfucT2genes were treated with the restriction enzyme SacI and were insertedinto the pEGW plasmid which had been treated with the same restrictionenzyme, to establish pEGWT(CO) (FIG. 1). FIG. 1 shows a metabolic routeto biosynthesize GDP-L-fucose and fucosyllactose in the Corynebacteriumglutamicum strain.

The gene sequences, strains, plasmids and oligonucleotides used in thepresent Example are shown in the following Tables 1 to 4.

TABLE 1 Genes and gene sequences Names of genes Sequence numbers manBSEQ ID NO: 1 manC SEQ ID NO: 2 gmd-wcaG SEQ ID NO: 3 fucT2 SEQ ID NO: 4lacYA SEQ ID NO: 5 COfucT2 SEQ ID NO: 6

TABLE 2 Strains Strains Related properties E. coli TOP10 F⁻, mcrAΔ(mrr-hsdRMS-mcrBC) f801acZΔM15 lacX74 recA1 araD139Δ (ara-leu) 7697galU galK rpsL (Str^(R)) endA1 nupG C. glutamicum Wild-type strain, ATCC13032

TABLE 3 Plasmids Plasmids Related properties pEKEx2 Km^(R); C.glutamicum/E. coli shuttle vector for regulated gene expression(P_(tac), lacIq, pBL1, oriVC.g., oriVE.c.) pVWEx2 Tc^(R); C.glutamicum/E. coli shuttle vector for regulated gene expression(P_(tac), lacIq, pHM1519, oriVC.g., oriVE.c.) pGRG36 Tn7 insertionvector, pSC101 replicon, Amp^(R) pBHA Cloning vector, pUC replicon,Amp^(R) pGlacYA pGRG36 + lacYA pBHA(COfucT2) pBHA + COfucT2 pEGWpEKEx2 + gmd-wcaG pVmBC pVWEx2 + manB + manC pEGWT pEGW + fucT2 pVBCLPVmBC + lacYA pEGWT(CO) pEGW + COfucT2

TABLE 4 Primers Primer Sequence names Sequence (5′→3′) numbersF_KpnI-gmd GGGGTACC AAGGAGATATACAATGT SEQ ID  CAAAAGTCGCTCTCATCACCNO: 7  R_SacI-wcaG CGAGCTCTTACCCCCGAAAGCGGTC SEQ ID  TTG NO: 8 F_PstI-manB AACTGCAG AAGGAGATATACAATGC SEQ ID  GTACCCGTGAATCTGTCACNO: 9  R_BamHI- CGGGATCCGGACTAGTGCTCTAGAT SEQ ID  SpeI-XbaI-TATGCGCGGATAATCCCTA NO: 10  manB F_XbaI-manC GCTCTAGA AAGGAGATATACAATGASEQ ID  CTTTAACTGACAAC NO: 11  R_SpeI-manC GGACTAGTCTACTGATCAGACGAAASEQ ID  A NO: 12  F_inf_ GTCCTTTTAACAGCGATCGCACCAT SEQ ID  AsiSI_lacYACGAATGGCGCAAAACCTTTCG NO: 13  R_inf_ GAGACGAAATACGCGATCGCGCTGT SEQ ID AsiSI_lacYA GGGTCAAAGAGGCATGATG NO: 14  F_inf_SacI_ GGGGGTAACTTAAGGAGCTCAAGGA SEQ ID  RBS_fucT2 GATATACAATGGCTTTTAAGGTGGT NO: 15  GCAAATTTGCGR_inf_SacI_ CGGCCAGTGAATTCGAGCTCTTAAG SEQ ID  fucT2CGTTATACTTTTGGGATTTTACCTC NO: 16  AAAATG F_SacI_RBS_ CGAGCTCAAGGAGATATACAATGG SEQ ID  COfucT2 NO: 17  R_SacI_CGAGCTCTTATGCGTTATACTTCTG SEQ ID  COfucT2 NO: 18  * Sequencesrepresented in italics mean RBSs (ribosome binding sites) and spacers. *Sequences represented in bold type mean recognition sites of specificrestriction enzymes

Example 2: Conditions and Methods for Culturing RecombinantCorynebacterium glutamicum

Seed culture was carried out using a test tube containing 5 mL of BHI(brain heart infusion) medium supplemented with an appropriateantibiotic (kanamycin 25 μg/mL, tetracycline 5 μg/mL) at a temperatureof 30° C. and a constant stirring rate of 250 rpm for 12 hours.

Batch culture was carried out at 30° C. in a 500 mL bioreactor(Kobiotech, Incheon, Korea) containing 100 mL or 1 L of minimum medium(containing (NH₄)₂SO₄ 20 g/L, urea 5 g/L, KH₂PO₄ 1 g/L, K₂HPO₄ 1 g/L,MgSO₄ 0.25 g/L, MOPS 42 g/L, CaCl₂ 10 mg/L, Biotin 0.2 mg/L,protocatechuic acid 30 mg/L, FeSO₄7H₂0 10 mg/L, MnSO₄H₂O 10 mg/L,ZnSO₄7H₂O 1 mg/L, CuSO₄ 0.2 mg/L, NiCl₂6H₂O 0.02 mg/L, pH 7.0). Thestirring rate during culture was maintained at 250 rpm for the flask and1000 rpm and 2 vvm for the bioreactor. In case of batch culture, IPTG(isopropyl-β-D-thiogalactopyranoside) and lactose were added such thatfinal concentrations were adjusted to 1.0 mM and 10 g/L, respectively,when optical density (OD₆₀₀) reached 0.8.

The fed-batch culture for high-concentration cell culture applicationwas carried out in a 2.5 L bioreactor (Kobiotech, Incheon, Korea)containing 1.0 L of a minimum medium supplemented with 40 g/L of glucoseand appropriate antibiotic (25 μg/mL of kanamycin, 5 μg/mL oftetracycline).

After the glucose added at an initial stage was completely consumed, afeeding solution including 800 g/L of glucose was supplied by acontinuous feeding method at a rate of 5.7 g/L/h. At the same time, IPTGand lactose were added such that final concentrations were adjusted to1.0 mM and 10 g/L, respectively in order to induce expression of tacpromotor-mediated genes and thereby produce 2′-fucosyllactose.

When pH of the medium was lower than a set point during fermentation,28% NH₄OH was automatically supplied and when pH was higher than the setpoint, 2N HCl was added, so that pH could be maintained within apredetermined range of (pH 6.98 to 7.02). The pH of the medium wasmeasured in real-time using a pH electrode (Mettler Toledo, USA).Stirring rate and aeration rate were maintained at 1,000 rpm and 2 vvmto prevent lack of oxygen.

Example 3: Determination of Concentrations of Cells and Metabolites

The dried cell weight was determined by multiplying the optical density(OD) by a pre-measured transmutation constant of 0.3. The opticaldensity (OD) was adjusted to the range of 0.1 to 0.5 by diluting asample at an appropriate level and absorbance at 600 nm was measuredusing a spectrophotometer (Ultrospec 2000, Amersham Pharmacia Biotech,USA).

The concentrations of 2′-fucosyllactose, lactose, lactate, glucose andacetic acid were measured using a high-performance liquid chromatography(HPLC) device (Agilent 1100LC, USA) equipped with a carbohydrateanalysis column (Rezex ROA-organic acid, Phenomenex, USA) and arefractive index (RI) detector. 20 μl of the culture medium diluted(×10) was analyzed using a column pre-heated at 60° C. 5 mM of a H₂SO₄solution was used as a mobile phase at a flow rate of 0.6 mL/min.

Test Example 1: Identification of Incorporation of lacZ Genes-RemovedLac Operon (lacYA) on Production of 2′-Fucosyllactose in Corynebacteriumglutamicum (C. glutamicum)

In the present Test Example, in order to bio-synthesize2′-fucosyllactose in Corynebacterium glutamicum (C. glutamicum), alactose carrier was incorporated and effects thereof were identified.

For this purpose, lac operon (lacYA), E. coli K-12-derived lacZ genes ofwhich are removed, established in the prior art (Korean Patent No.10-1544184), was incorporated into Corynebacterium glutamicum to produce2′-fucosyllactose.

In the prior art, in order to establish Escherichia coli that has noactivity of β-galactosidase and has only activity of the lactosecarrier, lac operon (lacYA) in which lac operon on the chromosome ofEscherichia coli is removed and lacZ genes encoding β-galactosidase areremoved is incorporated into the chromosome of Escherichia coli again,to produce 2′-fucosyllactose (Korean Patent No. 10-1544184).

Experimentation was conducted using the recombinant Corynebacteriumglutamicum strain that overexpressed only ManB, ManC, Gmd, and WcaG,which are GDP-L-fucose biosynthesis enzymes (Control Group), the strainthat overexpressed ManB, ManC, Gmd and WcaG, which are GDP-L-fucosebiosynthesis enzymes, and to which FucT2 was further incorporated(Comparative Example 1), and the strain in which lac operon (lacYA)obtained by removing ManB, ManC, Gmd, WcaG, FucT2 and lacZ genes, whichare GDP-L-fucose biosynthesis enzymes, was incorporated (Example 1). Bycomparing Control Group, Comparative Example 1 and Example 1 throughbatch culture in the flask, the effects of incorporation of lacYA operonand fucose transferase on production of 2′-fucosyllactose wereevaluated.

As a result of experimentation, the Corynebacterium glutamicum strainthat overexpressed only GDP-L-fucosebiosynthesis enzymes, ManB, ManC,Gmd and WcaG (Control Group), and the strain that overexpressed ManB,ManC, Gmd and WcaG, which are GDP-L-fucose biosynthesis enzymes, and inwhich FucT2 was further incorporated (Comparative Example 1), did notproduce 2′-fucosyllactose at all.

However, only the strain in which lac operon (lacYA) obtained byremoving ManB, ManC, Gmd, WcaG, FucT2 and lacZ genes, which areGDP-L-fucose biosynthesis enzymes, was incorporated (Example 1) produced2′-fucosyllactose (Table 5 and FIG. 2). FIG. 2 is a graph showingeffects of incorporation of lacZ-removed lac operon (lacYA) onproduction of 2′-fucosyllactose in Corynebacterium glutamicum. FIG. 2Ashows culture results of Control Group, FIG. 2B shows culture results ofComparative Example 1 and FIG. 2C shows culture results of Test Example1.

The results indicate that, as a lactose carrier is incorporated, lactoseis incorporated into the strain and is used to produce2′-fucosyllactose, which means that incorporation of lacZ genes-removedlac operon (lacYA) is essential for the production of 2′-fucosyllactose.

TABLE 5 Identification of effects of incorporation of lacZ-removed lacoperon (lacYA) on production of 2′-fucosyllactose in Corynebacterimnglutamicum (C. glutamicum) Yield Maximum 2′- (moles of 2′- Final driedLactose fucosyllactose fucosyllactose/ cell weight consumption^(a)concentration^(a) moles of Productivity^(a) Plasmid (g/L) (g/L) (mg/L)lactose) (mg/L/h) pVBC 13.5 0.02 N.D. — — pEGW pVBC 12.9 0.02 N.D. — —pEGWT pVBCL 13.4 0.78 246 0.22 5.0 pEGWT

Test Example 2: Production of 2′-fucosyllactose Through Batch Culture

In order to find the capability of the recombinant Corynebacteriumglutamicum (C. glutamicum) established in Test Example 1, to produce2′-fucosyllactose and fermentation features thereof, recombinantCorynebacterium glutamicum, in which lac operon (lacYA) from which ManB,ManC, Gmd, WcaG, FucT2 and lacZ were removed was incorporated wasbatch-cultured in a flask and a fermenter. IPTG and lactose were addedsuch that final concentrations were adjusted to 1.0 mM and 10 g/L,respectively, when the optical density (OD₆₀₀) reached 0.8.

As a result of flask batch culture, 246 mg/L of 2′-fucosyllactose wasproduced. The yield (ratio of moles of 2′-fucosyllactose to moles oflactose) was 0.22 mole/mole, and productivity was 4.97 mg/L/h (FIG. 3and Table 6).

Meanwhile, in case of fermenter batch culture, 274 mg/L of2′-fucosyllactose was produced, the yield (ratio of moles of2′-fucosyllactose to moles of lactose) was 0.34 mole/mole andproductivity was 5.6 mg/L/h. Compared to the flask culture, the finalconcentration, yield and productivity of 2′-fucosyllactose wereincreased by about 11%, 55% and 12%, respectively. This is due to thefact that the fermenter could efficiently control conditions such astemperature, pH and oxygen supply compared to the flask culture.

Results of the batch culture are shown in the following Table 6, FIG. 3is a graph showing results of batch culture using recombinantCorynebacterium glutamicum (C. glutamicum) pVBCL+pEGWT, FIG. 3A showsresults of flask batch culture and FIG. 3B shows results of fermenterbatch culture.

TABLE 6 Results of batch culture using recombinant Corynebacteriumglutamicum (C. glutamicum) pVBCL + pEGWT Yield Maximum 2′- (moles of 2′-Final dried Lactose fucosyllactose fucosyllactose/ cell weightconsumption^(a) concentration^(a) moles of Productivity^(a) (g/L) (g/L)(mg/L) lactose) (mg/L/h) Flask 13.4 0.78 246 0.22 4.97 Fermenter 13.00.57 274 0.34 5.6 ^(a)Concentrations of lactose and 2′-fucosyllactoseare calculated from only lactose and 2′-fucosyllactose present inmedium.

Test Example 3: Identification of Production of 2′-FucosyllactoseThrough LC-MS/MS Analysis

Qualitative analysis was conducted by LC-MS/MS in order to identify2′-fucosyllactose produced by batch culture of Test Example 2.

As a result of measurement of molecular weight in a cation mode usingMALDI-TOP MS, the peak of 511.164 m/z, which corresponds to themolecular weight of 2-fucosyllactose having one sodium molecule bondedthereto was observed.

In addition, as a result of tandem mass spectrometry (MS/MS) analysis toidentify the structural composition of the peak, glucose, galactose andfucose that constitute 2′-fucosyllactose were found (FIG. 4). FIG. 4shows results identifying production of 2′-fucosyllactose throughLC-MS/MS analysis of the culture solution of recombinant Corynebacteriumglutamicum. FIG. 4A shows results identifying production offucosyllactose through molecular weight analysis of ingredientscontained in the culture solution in a cation mode using MALDI-TOP MS,and FIG. 4B shows tandem mass spectrometry (MS/MS) results structurallyidentifying the fact that the peak of 511.134 m/z corresponds to2′-fucosyllactose.

As a result of experimentation, 2′-fucosyllactose was produced in thebatch culture.

Test Example 4: Production of 2′-fucosyllactose Through Fed-BatchCulture

In order to produce high-concentration 2′-fucosyllactose throughhigh-concentration cell culture, fed-batch culture was conducted in a2.5 L fermenter using recombinant Corynebacterium glutamicum (C.glutamicum) in which pVBCL and pEGWT plasmids were incorporated.

When 40 g/L glucose supplied at an initial stage was completelyconsumed, a feeding solution started to be supplied at a rate of 5.7g/L/h by a continuous feeding method in order to maintain cell growth.At the same time, IPTG and lactose were added to induce production of2′-fucosyllactose.

As a result of experimentation, acetic acid was not produced at allduring fermentation, and the cells had a dried cell weight of 57.3 g/Lthrough metabolism of glucose. In addition, maximum concentration of2′-fucosyllactose was 5.8 g/L, the yield (ratio of moles of2′-fucosyllactose to moles of lactose) was 0.55 mole/mole andproductivity was 0.06 g/L/h (FIG. 5 and Table 7).

Results of fed-batch culture to produce 2′-fucosyllactose are shown inthe following Table 7, and FIG. 5 is a graph showing results offed-batch culture using recombinant Corynebacterium glutamicumpVBCL+pEGWT.

TABLE 7 Results of fed-batch culture using recombinant Corynebacteriumglutamicum (C. glutamicum) pVBCL + pEGWT Yield Maximum 2′- (moles of 2′-Final dried Lactose fucosyllactose fucosyllactose/ cell weightconsumption^(a) concentration^(a) moles of Productivity^(a) Plasmid(g/L) (g/L) (g/L) lactose) (g/L/h) pVBCL 57.3 7.3 5.8 0.55 0.06 pEGWT^(a)2-FL productivity was calculated after IPTG induction.^(b)Concentrations of lactose and 2′-fucosyllactose are calculated fromonly lactose and 2′-fucosyllactose present in medium.

[Test Example 5: Effects of Incorporation of Codon-Optimized fucT2 Genes(COfucT2) on Production of 2′-Fucosyllactose by RecombinantCorynebacterium glutamicum (C. glutamicum)]

(1) Production of Codon-Optimized fucT2 Genes (COfucT2)

In order to improve translation efficiency of fucT2 genes which areHelicobacter pylori (H. pylori)-derived α-1,2-fucosetransferase inrecombinant Corynebacterium glutamicum (C. glutamicum), the fucT2 geneswere codon-optimized depending on codon usage of Corynebacteriumglutamicum.

As a result of experimentation, when compared with conventional fucT2genes, about 17.1% of the sequence was mutated (FIG. 6). FIG. 6 showsresults of codon-optimization of fucT2 genes to be suited toCorynebacterium glutamicum (C. glutamicum).

(2) Identification of Effects of Incorporation of Codon-Optimized fucT2Genes (COfucT2) on Production of 2′-Fucosyllactose

In order to find the capability of the recombinant Corynebacteriumglutamicum (C. glutamicum) established using codon-optimized fucT2 genes(COfucT2) to produce 2′-fucosyllactose, and fermentation featuresthereof, batch culture and fed-batch culture were carried out in a flaskand a fermenter, respectively.

In case of batch culture, IPTG and lactose were added such that finalconcentrations were adjusted to 1.0 mM and 10 g/L, respectively, whenthe optical density (OD₆₀₀) reached 0.8. In case of fed-batch culture,when 40 g/L glucose supplied at an initial stage was completelyconsumed, a feeding solution started to be supplied at a rate of 5.7g/L/h by a continuous feeding method to maintain cell growth. At thesame time, IPTG and lactose were added to induce production of2′-fucosyllactose.

As a result of batch culture, 370 mg/L of 2′-fucosyllactose wasproduced, the yield (ratio of moles of 2′-fucosyllactose to moles oflactose) was 0.28 mole/mole and productivity was 7.18 mg/L/h (FIG. 7Aand Table 8). Compared to results of Test Example 2, the finalconcentration, yield and productivity of 2′-fucosyllactose wereincreased by about 50%, 27% and 44%, respectively.

Meanwhile, as a result of fed-batch culture, 8.1 g/L of2′-fucosyllactose was produced, the yield (ratio of moles of2′-fucosyllactose to moles of lactose) was 0.42 mole/mole andproductivity was 0.07 g/L/h (FIG. 7B and Table 8). Compared to resultswhen wild-type fucT2 was incorporated (Test Example 4), the finalconcentration and productivity of 2′-fucosyllactose were increased byabout 39% and 17%, respectively.

The results of culture are shown in the following Table 8, FIG. 7A is agraph showing results of flask batch culture using recombinantCorynebacterium glutamicum (C. glutamicum) pVBCL+pEGWT (CO), and FIG. 7Bis a graph showing results of fermenter fed-batch culture usingrecombinant Corynebacterium glutamicum (C. glutamicum) pVBCL+pEGWT(CO).

TABLE 8 Results of batch and fed-batch culture using recombinantCorynebacterium glutamicum (C. glutamicum) pVBCL + pEGWT(CO) Yield(moles of 2′- Final dried Lactose Maximum 2′- fucosyllactose/ cellweight consumption^(a) fucosyllactose moles of (g/L) (g/L)concentration^(a) lactose) Productivity^(a) Batch 14.2 0.94 370 (mg/L)0.28 7.18 (mg/L/h) (flask) Fed-batch 62.1 13.6 8.1 (g/L) 0.42 0.07(g/L/h) (fermenter) ^(a)2-FL productivity was calculated after IPTGinduction. ^(b)Concentrations of lactose and 2′-fucosyllactose werecalculated from only lactose and 2′-fucosyllactose present in medium.

The invention claimed is:
 1. A method of producing 2′-fucosyllactosecomprising culturing in a medium supplemented with lactose a recombinantCorynebacterium glutamicum transformed to expressα-1,2-fucosyltransferase, transformed to expressGDP-Dmannose-4,6-dehydratase, transformed to express GDP-L-fucosesynthase, and transformed to express lactose permease, and isolating2′-fucosyllactose from the medium, wherein the recombinantCorynebacterium glutamicum has phosphomannomutase andGTP-mannose-1-phosphate guanylyltransferase.
 2. The method according toclaim 1, wherein the medium further comprises glucose.
 3. The methodaccording to claim 2, wherein the production of the fucosyllactose iscarried out by fed-batch culture comprising further supplying glucose orlactose.
 4. The method according to claim 1, wherein the recombinantCorynebacterium glutamicum is transformed to overexpressphosphomannomutase, and transformed to overexpressGTP-mannose-1-phosphate guanylyltransferase.